Archivum Immunologiae et Therapiae Experimentalis (2020) 68:25 https://doi.org/10.1007/s00005-020-00585-3

REVIEW

Cathepsin G and its Dichotomous Role in Modulating Levels of MHC Class I Molecules

Timo Burster1 · Uwe Knippschild2 · Ferdinand Molnár1 · Anuar Zhanapiya1

Received: 30 November 2019 / Accepted: 11 June 2020 © L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland 2020

Abstract G (CatG) is involved in controlling numerous processes of the innate and adaptive immune system. These features include the proteolytic activity of CatG and play a pivotal role in alteration of chemokines as well as cytokines, clearance of exogenous and internalized pathogens, platelet activation, apoptosis, and antigen processing. This is in contrast to the capability of CatG acting in a proteolytic-independent manner due to the net charge of arginine residues in the CatG sequence which interferes with bacteria. CatG is a double-edged sword; CatG is also responsible in pathophysiological conditions, such as autoimmunity, chronic pulmonary diseases, HIV infection, tumor progression and metastasis, photo-aged human skin, Papillon–Lefèvre syndrome, and chronic infammatory pain. Here, we summarize the latest fndings for functional responsibilities of CatG in immunity, including bivalent regulation of major histocompatibility complex class I molecules, which underscore an additional novel role of CatG within the immune system.

Keywords · · T regulatory cells · NK cells · MHC · Lactoferrin · -activated receptor

Abbreviations TGF-β Transforming growth factor beta APCs Antigen-presenting cells Th T helper Cat Cathepsin Tregs T regulatory cells CatG Cathepsin G Tregs Thymus-derived natural Tregs cDCs Conventional dendritic cells T1D Type 1 diabetes mellitus DCs Dendritic cells HIV Human immunodefciency virus HLA Human leukocyte antigen Introduction LF Lactoferrin MHC Major histocompatibility complex Proteases are proteolytic that participate in physi- NE Neutrophil ological cell metabolism and are mainly responsible for NETs Neutrophil extracellular traps protein degradation by hydrolysis. As a consequence, they NK Natural killer are functional in the immune system, for instance, for pro- PAR Protease-activated receptor cessing of intracellular antigens within antigen-presenting PBMCs Peripheral blood mononuclear cells cells (APCs) (Kramer et al. 2017; Sadeghzadeh et al. 2020). PMSF Phenylmethylsulfonyl fuoride Neutrophils, recruited and activated at the site of infamma- SDF1 Stromal cell-derived factor 1 tion, naturally release diferent classes of mediators to the TCR T cell receptor surrounding environment and thereby support an immune response. One example is serine proteases, such as cathep- sin G (CatG), protease 3 (PR3), (NE), * Timo Burster [email protected] and neutrophil resident in primary granules compared to lactoferrin (LF) located in secondary granules. 1 Department of Biology, School of Sciences and Humanities, All of them harbor the serine amino acid residue at the active Nazarbayev University, Kabanbay Batyr Ave. 53, center, which is the reason for their classifcation. Serine Nur‑Sultan 010000, Kazakhstan proteases comprise a common feature of structural proper- 2 Department of General and Visceral Surgery, Surgery Center, ties of the active center with a consisting of Ulm University Hospital, 89081 Ulm, Germany

Vol.:(0123456789)1 3 25 Page 2 of 13 Archivum Immunologiae et Therapiae Experimentalis (2020) 68:25 histidine, aspartate, and serine amino acids, which allows act in a proteolytic-dependent and -independent antimicro- the peptide bound between amino acids within the protein bial manner through the positive net charge of CatG, which sequence to be hydrolyzed (AhYoung et al. 2019; Grzywa is due to arginine residues within the CatG sequence. et al. 2019; Korkmaz et al. 2018). The proteolytic milieu Furthermore, neutrophils can release extracellular traps is critical for the integrity and function of the efective (neutrophil extracellular traps: NETs), enclosing DNA immune system. However, to avoid unintended and con- and histones as well as cytoplasmic granular proteins, like tinuous proteolytic degradation, immune cells express ser- CatG, to catch and trap microorganisms in the extracellular ine protease inhibitors (serpins) which in turn inhibit serine space, since CatG has a high afnity to deoxyribonucleic protease activity (Burgener et al. 2019; Cooley et al. 2001), acid (Liu and Liu 2020; Ma et al. 2019). However, CatG a mechanism to prevent damage of healthy cells or to block is involved in autoimmunity, chronic pulmonary diseases, the destruction of the neighboring tissue. These inhibitors human immunodefciency virus (HIV) infection, tumor are also essential in the advanced life cycle of cells and the progression, and metastasis (Burster 2013; Meyer-Hofert process of aging, since the proteolytic activity of proteases 2009; Pham 2008). Furthermore, it was shown that CatG under such circumstances is not properly regulated (Groma- is increased in photo-aged human skin and primary fbro- kova and Konovalenko 2003; Vaughan-Thomas et al. 2000; blasts (Zheng et al. 2012); interestingly, specifc inhibition Wyczalkowska-Tomasik and Paczek 2012). Proteases are not of CatG prevents ultraviolet B-induced photo-aging (Son only involved in physiological function, they have the ability et al. 2012). From a clinical point of view, the absence to act under pathophysiology conditions. For instance, high of active CatG, protease three, and neutrophil elastase in proteolytic activity of serine proteases in cancer stem cells activated neutrophils results in the reduced ability of neu- supports tumor development (Hillebrand and Reinheckel trophils in phagocytosis and NET formation to neutralize 2019) and CatG is increased in acute lymphoid leukemia microorganisms. Increased generation of reactive oxygen (Khan et al. 2018), indicating a logical rational for apply- species and release of infammatory mediators underly- ing pharmacological protease inhibitors to prevent tumori- ing host tissue damage and is responsible for the outcome genesis, which certainly may not interfere with the general of Papillon–Lefèvre syndrome, which is characterized by proteolytic responsibility of the protease in the organism. periodontitis followed by a progressive loss of teeth at a are lysosomal/endosomal proteases respon- young age. A reason for that lays in a mutation in the cath- sible for protein degradation within the endocytic com- epsin C which inactivates the proteolytic capacity of partment. A majority of these proteases are involved in and is thereby not able to convert serine pro- generating antigenic peptides to be loaded into the peptide teases to their active form (Korkmaz et al. 2018; Sorensen binding groove of major histocompatibility complex class et al. 2014). As previously reported, CatG is signifcantly II (MHC II) molecules and presented to T helper (Th) cells. upregulated in chronic infammatory pain and inhibition In addition, cathepsins are found in the nucleus or nuclear of CatG reduced pain behavior by infltrating neutrophils membrane, on the cell membrane, and extracellular space in rodents (Liu et al. 2015). Moreover, increased levels of in order to activate or deactivate protein- or peptide-based CatG correlate with neutrophil counts in synovial fuid and mediators by hydrolysis. Cathepsins belong to three diferent in rheumatoid arthritis tissue, in contrast CatG-defcient mechanistic classes of proteases, as noted in the previous mice as well as mice defcient in CatC are resistant to section CatG is a serine protease, whereby cathepsin A is experimental arthritis (Adkison et al. 2002; Hu and Pham also referred to this class, compared to cathepsins B, C, F, 2005; Miyata et al. 2007). On the one hand, CatG plays a H, K, L, O, S, V, W, and X which are classifed as cysteine signifcant role in protecting and defending the organism proteases. The third class are aspartic proteases, such as from invading pathogens; on the other hand, depending cathepsins D and E, as has been extensively reviewed in on its proteolytic activity, CatG can cause harm when it Bischof et al. (2017), Cresswell (2019), Holzen et al. (2020) is active over an extended period of time. Therefore, dur- and Zhang et al. (2020). ing infammation, CatG harbors benefcial efects to fght In this briefng, we will focus on the signifcance of infections, but after a successful immune response, CatG CatG with respect to its functional role in the immune sys- activity has to be tightly regulated to a steady state level. tem. The proteolytic activity of CatG modifes chemokines In the next sections, we take our recent fndings into and cytokines, clearance of internalized pathogens, activa- account to elucidate the function of CatG during an tion of cell surface receptors, platelet activation, apopto- immune response. This includes how LF facilitates the sis, and antigen processing (Burster 2013; Fu et al. 2020; performance of CatG and insight into how CatG regulates Zamolodchikova et al. 2020). CatG provokes proliferation intracellular and extracellular cell surface levels of MHC I of ­CD4+ T cells, and to a lesser extent B cells but not molecules, which indicates a novel but dichotomous char- ­CD8+ T cells (Hase-Yamazaki and Aoki 1995). CatG can acteristic of CatG within the immune system.

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Regulation of CatG Activity and Functional peptide bonds after R and K amino acid residues), chymo- Consequences in Regulation of MHC (F, Y, and W), metase (M), and leuase (L) cleavage Molecules properties at the P1 subsite within the substrate; however, mouse CatG is limited to -like activity (F, Y, MHC: The Two Classes and W) (Raymond et al. 2010). Strikingly, an additional cleavage site at the P1 position harboring asparagine (N), Professional APCs [macrophages, B cells, and dendritic P2 proline (P), and for P3 position glutamic acid (E) was cells (DCs)], granulocytes, natural killer (NK) cells, cyto- determined recently by high throughput protease profling toxic T lymphocytes (CD8­ + T cells), and Th cells (CD4­ + T (Nguyen et al. 2018). Focusing on the C-terminal end within cells) represent the majority of immune cells of the immune a given peptide, human CatG has a cleavage preference for system. ­CD8+ T cells recognize antigenic peptides properly isoleucine (I), alanine (A), and serine (S) at P1′ and nega- loaded to MHC I molecules, which are expressed by almost tively charged amino acids (D and E) are preferred at the all cells of the organism; whereas, antigenic peptides loaded P2′ position as was observed in an extended study (Nguyen to MHC II molecules and presented by professional APCs et al. 2018; Thorpe et al. 2018). According to the three- are engaged and inspected by ­CD4+ T cells (Jurewicz and dimensional structure, CatG has two homologous β-barrels Stern 2019). with six antiparallel β-sheets joined with a linker segment for each barrel motif (Fig. 1). The catalytic triad of H­ 57, ­D102, and S­ 195 is located between the two-barrel folding pat- Modulation of CatG Activity tern, where the active-site cleft is perpendicular to the two β-barrels allowing the hydroxyl group of ­S195 to attack the It is well known that the 235-amino acid covering polypep- carbonyl carbon of the peptide bond (substrate) in a nucleo- tide CatG (~ 28 kDa), which was isolated in 1976 from the philic manner (Hof et al. 1996; Korkmaz et al. 2010). In human spleen, is highly active at a neutral pH, and exhibits humans, four polymorphisms in the 5′-fanking region with chymotrypsin—and trypsin-like enzymatic activity (Pow- no efects regarding the promotor- or transcription activ- ers et al. 1985; Starkey and Barrett 1976a, b). Surely, this is ity were observed, opposed to the polymorphism found in quite simplifed since human CatG has trypsin (hydrolyzes the coding region (N­ 125S), which is correlated with a lower

Fig. 1 Three-dimensional structure of human CatG (ribbon plot). The N-linked glycosylation site is found at N­ 65 and the polymorphism in catalytic triad of CatG is indicated by H­ 57, ­D102, and S­ 195 which is the coding region of CatG is labeled as ­N119S. The inhibitor Suc-Val- P located between the two asymmetric barrels and depicted in yellow. Pro-Phe -(OPh)2 (violet stick model) is complexed with CatG. PDB: The C-terminal α-helix is shown on top and presented in blue. The 1CGH (Hof et al. 1996)

1 3 25 Page 4 of 13 Archivum Immunologiae et Therapiae Experimentalis (2020) 68:25 survival rate after a cardiovascular or cerebrovascular epi- during inflammation or wound healing, since it is well sode due to high levels of plasma fbrinogen in patients known that CatG activates platelets via protease-activated with ­N125S polymorphism (Herrmann et al. 2001). The sin- receptors (PARs) (Heuberger and Schuepbach 2019; Selak gle N-linked glycosylation site at N­ 71 categorizes CatG for et al. 1988) and an LF-mediated increase of CatG activity three diferent glycosylation isoforms, while the fourth iso- further activates platelets as demonstrated by us just recently form is not attached to a carbohydrate side chain (Loke et al. (Eipper et al. 2016). Moreover, platelets can coat tumor cells 2015; Salvesen et al. 1987; Watorek et al. 1993). Moreover, and transfer their MHC I content to tumor cells in such a way it was shown that a monocyte cell line (U937) segregated that NK cells are not activated when tumor cells reduce lev- the ~ 32 kDa proCatG to the extracellular environment and els of MHC I and hide tumor-associated peptide presentation the ~ 28 kDa CatG form resides in the lysosome (Lindmark on the tumor cell surface and thereby support an immune et al. 1990, 1994). In addition, the diverse glycosylation sta- evasion of tumor cells (Erpenbeck and Schon 2010; Placke tus can destine CatG for secretion or lysosomal storage as et al. 2012). Based on these fndings, it can be speculated suggested by Watorek et al. (1993). that CatG is involved in platelet-mediated tumor progres- Activated polymorphonuclear neutrophils release LF as sion, since activated neutrophils secrete CatG and LF, which well as CatG and other serine proteases during an immune support the performance of platelets, indicating a dual role response (Korkmaz et al. 2010); therefore, engagement of of CatG. LF with CatG was investigated and determined that LF is As we saw in the previous section that LF can increase a natural inhibitor of CatG (He et al. 2003). Having this the catalytic activity of CatG, further components, such as in mind, we recently investigated the molecular interaction 1,25 (OH)2D3 (vitamin D), have the potential to modulate between LF and CatG. LF was incubated with CatG and immune cells. Indeed, vitamin D provokes a reduction of the catalytic activity of CatG was determined by active-site DCs in the ability to prime T cells (Adorini 2003), sug- label and kinetics; however, we found an increase of gesting a therapeutic potential of vitamin D for treatment CatG activity by LF, proposing that LF interacts with CatG of autoimmunity since auto-aggressive T cells with the in an allosteric manner (Eipper et al. 2016). These fndings potential to destroy healthy cells or tissues are inhibited. were verifed by an independent group to obtain a better Consistent with these fndings, it has been revealed that the understanding of the enhancing efect by using a protein expression and activity of CatG are elevated in type 1 dia- digestion assay. Thorpe et al. (2018) found an increase of betes mellitus (T1D)-derived peripheral blood mononuclear protein (thioredoxin) turnover rate when an excess of the cells (PBMCs), and inhibition of CatG by a specifc inhibitor LF protein was incubated with CatG, even though a specifc, or by using vitamin D reduce the activation of proinsulin- direct interaction of LF with CatG might be unlikely. Inter- reactive, so called diabetogenic, T cells. Moreover, freshly estingly, levels of LF was found to be high and the catalytic isolated conventional DCs (cDCs) from the blood of healthy activity of CatG was signifcantly increased in serum from donors treated with vitamin D showed a reduction of CatG osteomyelitis patients consisting of ­N125S polymorphism in activity determined by the active-site label (Zou et al. 2011). contrast to osteomyelitis patients with the ­N125 phenotype. A recent study has suggested that vitamin D is responsible These fndings of increased CatG activity can be explained for upregulation of serine protease inhibitors in ­CD11c+ by the interaction of LF and CatG, but more likely by the bone marrow-derived mouse DCs (Saul et al. 2019), which change of ­N125S within the CatG protein sequence (Perez-Is gives rise to the speculation that these fndings explain why et al. 2019). vitamin D decreases CatG activity in cDCs (Zou et al. 2011). Furthermore, LF broadens the substrate cleavage prefer- Interestingly, cDCs from T1D patients did not show any dif- ence of CatG by lowering the substrate selectivity even for ference in CatG activity when cDCs were treated with vita- valine, based on the fact that CatG does not normally accept min D, in contrast to cDCs from healthy individuals (Zou valine at the P1 position (Eipper et al. 2016). The functional et al. 2011). As a result, altered CatG activity has functional consequences can be that LF directs and alters CatG activ- consequences during an immune response. ity for a more efcient performance of degrading patho- gen-derived proteins due to the acceptance of an increased Intracellular Proteolytic Regulation of MHC I number of diferent amino acid residues at P1. Moreover, invading bacteria lower the pH environment through anaero- Endocytic proteases, cathepsins, cleave proteins into smaller bic metabolism (Menkin 1956). Interestingly, LF recovers oligopeptides, and amino acids, thereby controlling the CatG activity even at a lower pH (pH 5) indicating that LF proteome steady state in the cell. The endocytic compart- avoids reduction of CatG activity at the site of infammation ments within APCs contain cysteine, aspartic, and serine in order to still be able to fght against pathogens (Eipper proteases, the concerted action of which generates peptides et al. 2016). Another reason for an LF-mediated increase of from self and foreign antigens to be loaded to MHC II mol- CatG activity might be to support the function of platelets ecules. There is crosstalk (cross-presentation) between the

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MHC II and MHC I antigen processing pathways (Colbert TAP-independent MHC I cross-presentation in vivo. More- et al. 2020). It is worth noting that cathepsins along with over, MHC I molecules can reach the phagosome, where other proteolytic enzymes cooperate with the proteasome MHC I molecules are loaded with exogenous/endocytic- with regard to antigen processing and generating antigenic derived antigenic peptides and the MHC I-peptide complex peptides for antigen presentation via MHC I (Jurewicz and appears at the cell surface for inspection by patrolling CD8­ + Stern 2019). T cells (Gromme et al. 1999; Grotzke et al. 2017; Hochman APCs accommodate CatG. These include primary B et al. 1991; Reid and Watts 1990; Schirmbeck et al. 1995; cells, monocytes, plasmacytoid DCs, cDCs, cortical thymic Shen et al. 2004). MHC I molecules passing the recycling epithelial cells, murine microglia, and macrophages; how- pathway or the phagosome without traveling to the cell ever, CatG is absent in B lymphoblastoid cell lines or surface possibly degraded in the lysosome. In this regard, monocyte-derived DCs. Interestingly, CatG non-expressing we addressed the question of which protease is crucial in cell lines can take up CatG from the surrounding envi- degrading MHC I molecules and found that CatG, D, and S ronment to expand their intracellular protease repertoire proteolytically degrade MHC I molecules in vitro (Palesch for antigen processing (Burster et al. 2004; Stoeckle et al. et al. 2016). However, the treatment of PBMCs with selec- 2009). Beyond antigen processing, further functional roles tive inhibitors for CatG, D, and S, provoked an increase in of CatG were determined by incubating MHC II molecules levels of MHC I molecules only when PBMCs were treated with CatG. CatG cleaves MHC II molecules in vitro but the with the CatG inhibitor. Moreover, introduction of CatG in CatG cleavage site is sterically inaccessible when MHC II glioblastoma stem cells and human embryonic kidney 293 molecules are integrated into the membrane (Burster et al. cells leads to a signifcant downregulation of cell surface 2010). Thus, MHC II molecules are stable and resistant to MHC I molecules, since MHC I molecules are proteolyti- CatG-mediated degradation in the endocytic compartment, cally digested by endogenous CatG resident in the endo- which is important for antigenic peptide loading on MHC II cytic compartment, which might be an efective approach molecules and being transferred to the cell surface to display to sensitizing NK cells to target cancer cells (Palesch et al. the intracellular peptide status. The question remains as to 2016) (Fig. 2). whether solubilized MHC I molecules are also proteolytic One immune evasion mechanism of cancer cells includes hydrolyzed and whether MHC I molecules resist degradation the downmodulation of cell surface MHC I molecules or the when integrated into the membrane. intracellular arrest of MHC I molecules to avoid recognition The MHC I molecule is expressed by a heavy chain and by CD8­ + T cells, after which cancer cells instantly maintain β2-microglobulin on the cell surface. In humans, MHC I a limited set of MHC I molecules and escape destruction molecules are encoded by the three classical loci known as by NK cells (Paul and Lal 2017). Viruses have developed human leukocyte antigen (HLA)-A, HLA-B, and HLA-C. a similar strategy to hold back MHC I molecules from the The classical pathway of MHC I-restricted antigen presenta- cell surface, for instance, HIV-1 prevents HIV-infected cells tion starts with cytosolic antigen processing generally per- from displaying MHC I loaded with HIV-derived antigenic formed by the proteasome-ubiquitin system and transported peptides to avoid CD8­ + T cell-mediated killing. Two models into the endoplasmic reticulum (ER) lumen. It is here that were proposed: At early infection (around 48 h) the HIV- antigenic peptides are loaded to MHC I molecules with the 1-derived accessory protein Nef protein forces MHC I mole- help of the transporter associated with antigen processing cules to be endocytosed. When the infection progresses, Nef (TAP), tapasin, calreticulin, and ERp57; this MHC I–peptide interferes with the transport of nascent MHC I molecules to complex trafcs to the cell surface to be inspected by ­CD8+ the cell surface (Dikeakos et al. 2010; Schwartz et al. 1996). T cells. The presence of peptide-loaded MHC I molecules In the case of the endocytosis route, Nef binds to the cyto- on the cell surface can provoke ­CD8+ T cell activation when plasmatic tail of MHC I during endocytosis. Nef:MHC I recognizing, for instance, pathogen-derived antigenic pep- transits from the early endosome to the late endosome and tides (Santambrogio and Rammensee 2019). fnally reaches the trans-Golgi network where Nef:MHC Besides the classical MHC I antigen processing and pres- I molecules accumulate. Strikingly, in these experiments, entation pathway, cross-presentation of exogenous antigens Nef:MHC was not detected in lysosomes (Dirk et al. 2016), is possible in DCs, B cells, and macrophages. Eventually, which challenges the general statement that Nef:MHC I tar- surface MHC I molecules are endocytosed to undergo a new gets to the lysosomal compartment for degradation (Pereira round of antigen loading in the endocytic compartment, and and daSilva 2016). It is important to note that Nef down- transit back to the cell surface (recycling pathway). Another modulates, but not completely, HLA-A, HLA-B, and the pathway comprises the trafcking of MHC I molecules non-classical MHC molecule HLA-E (Apps et al. 2015; van from the ER via the trans-Golgi network to the endocytic Stigt Thans et al. 2019); however, HLA-C cell surface levels compartment (vacuolar pathway). In this pathway, cath- are reduced by the viral protein U (Vpu; Vpu has a similar epsin S plays an important role in generating peptides for function like Nef) from most primary HIV-1 strains (Apps

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◂Fig. 2 CatG-mediated intracellular proteolytic regulation of MHC I. added to the assay increases cell surface density of MHC a In general, immune cells express CatG or can take up CatG from I molecules of the respective target cell, but also PBMCs, the surrounding environment. Cell surface MHC I molecules passing the recycling pathway are degraded by resident CatG in the endo- which carry CatG on the cell surface, trigger enhancement some (or lysosome). b Expression of MHC I molecules is impaired of MHC I expression on the cell surface of a monocytic in glioblastoma cells (immune evasion). CatG is absent in glioblas- cell line (THP-1) (Giese et al. 2016). The reason for this toma cells; however, introduction of CatG in glioblastoma cells pro- could be that CatG bound on the cell surface of immune vokes a signifcant downregulation of cell surface MHC I molecules. CatG digest MHC I molecules, most likely in the endosome. In this cells provokes directly, or by soluble CatG secreted at the scenario, reduced cell surface MHC I molecules might sensitize NK site of infammation by activated neutrophils, an increase of cells to target cancer cells cell surface MHC I to display the intracellular antigen status of the target cell which can be monitored by ­CD8+ T cells. Some viral strains can hold back nascent MHC I molecules et al. 2016). Whether MHC I molecules during tracking in to remain in the cell. Subsequently, antigen-specifc ­CD8+ T the endocytic compartments (and eventually reach the trans- cells are not activated and viral infected cells evade immune Golgi network) are proteolytically degraded by CatG needs detection. to be experimentally validated. CatG is found intracellularly (Schroeder et al. 2020) but It has been previously shown that membrane-associated not on the cell surface of resting ­CD8+ T cells (Delgado RING-CH-type fnger 4 (MARCH4) protein and MARCH9 et al. 2001; Yamazaki and Aoki 1997), in contrast to double promote the downregulation of cell surface MHC I mol- positive CD4­ +CD8+ T cells (Penczek and Burster 2019). ecules by triggering MHC I ubiquitination and subsequently CD4­ +CD8+ T cells show cytotoxic as well as a suppres- forcing lysosome degradation (Bartee et al. 2004). Recent sive capacity depending on the conditions in which they findings suggest that endogenous MARCH9 mediates are present. In the case of cytotoxicity towards infection MHC I ubiquitination and facilitates delivery of nascent or to recognizing cancer cells, the T cell receptor (TCR) synthesized MHC I molecules loaded with peptides from of CD4­ +CD8+ T cells interact with MHC I molecules the trans-Golgi network into the endocytic compartment (Overgaard et al. 2015). Therefore, it is most likely that for efcient antigen cross-presentation in response to spe- ­CD4+CD8+ T cells directly have the possibility to induce an cifc environmental signals (De Angelis Rigotti et al. 2017). increase of MHC I via CatG to monitor the antigenic peptide Taken together, endogenous CatG might digest ubiquitin- repertoire and are able to kill infected cells. This hypothesis labeled MHC I molecules and serve as an essential protease and whether viral strains can prevent (or not) CatG-mediated for recycling and post-transcriptional regulation of MHC I nascent MHC I from reaching the cell surface and whether molecules. the viral-derived antigenic peptidome might change during this process remains to be proven experimentally. Extracellular CatG and its Functional Role The question arises as to why CatG is absent on the cell in Controlling Cell Surface Expression of MHC I surface of ­CD8+ T cells (Delgado et al. 2001; Yamazaki and Aoki 1997). A previous study showed that CatG can bind By screening the cell surface of diferent immune cells, it to ­CD4+ T cells, NK cells, and B cells, in addition to ­CD8+ was found that CatG is bound on the cell surface of several T cells (Yamazaki and Aoki 1997). During infammation, cells of the immune system. Such cells contain neutrophils where CatG is nearby, ­CD8+ T cells can bind CatG and push (Owen et al. 1995), B cells, NK cells (Delgado et al. 2001), out intracellular MHC I of the target cell. In this scenario, and platelets (Selak 1994), whereas CatG is only poorly viral-derived antigenic peptides can be displayed on MHC I expressed on ­CD4+ T cells (Delgado et al. 2001). It remains and recognized by antigen specifc TCR of CD8­ + T cells. In an open question as to functional role of CatG on the cell contrast, when CatG is absent on ­CD8+ T cells, these cells surface. might represent, most likely, resting ­CD8+ T cells. Another To compare levels of MHC I on DCs from CatG-defcient reasonable idea could be that CatG bound on the cell surface mice and wild type, we found that DCs from CatG-defcient of ­CD8+ T cells has the capability to protect activated ­CD8+ mice express less cell surface MHC I molecules in contrast T cells from secreted perforin and B and avoids to their wild-type counterparts (Giese et al. 2016). This is self-destruction (similar to NK cells, discussed below). due to the action of CatG, since exogenous CatG facilitates The absence or at least decline of MHC molecules, the upregulation of cell surface MHC I molecules on PBMCs predominantly detected in tumor or virus-infected cells, and human glioblastoma cells, most likely via PAR1, as can provoke NK cell activation by secretion of cytokines/ summarized in Fig. 3. Furthermore, LF, which increases chemokines, , and perforins for target cell kill- the proteolytic activity of CatG, serves as an enhancer of ing purposes (Moretta et al. 2016). Recently, our group exogenous CatG-induced upregulation of MHC I molecules has shown that two NK cell subsets ­(CD16–CD56dim and on PBMCs and on a B cell line. Not only purifed CatG CD16­ dimCD56−) have active CatG on the cell surface

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which is absent on ­CD16–CD56bright, ­CD16dimCD56bright, activity-based probe MARS116 and avidin-FITC was estab- ­CD16brightCD56dim, ­CD16dimCD56dim, and ­CD16brightCD56−. lished to determine the proteolytic activity of CatG on the For these experiments, an active-site label using the cell surface of distinct NK cell subsets (Penczek et al. 2016).

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◂Fig. 3 A model of how extracellular CatG controls cell surface Further experiments are necessary to reveal the functional expression of MHC I. a PAR1 belongs to a family of G protein-cou- purposes of CatG on ­CD39+ Tregs. One might speculate pled receptors activated by proteases. CatG proteolytically cleaves the N-terminal end of the extracellular domain of PAR1; thereby the teth- that cell surface CatG might also force MHC I upregula- ered activation ligand fips to the extracellular loop two and recruits tion or CatG to protect Tregs, which can exocytose perforin intracellular G protein. G protein is capable of downstream signal (and ) for cytolysis of efector cells (Grossman transsduction, which results in an efector function, for instance, an et al. 2004) from self-destruction by proteolytic processing infammatory response. b LF increases the proteolytic activity of + CatG. CatG cleavage of the extracellular part of PAR1 can also lead of perforin and granzyme B, similar to NK cells or ­CD8 to receptor inactivation (“dis-arming”) by degrading the tethered T cells. Moreover, the growth and maturation factor CXC ligand as well as three extracellular loops of PAR1. This process chemokine stromal cell-derived factor 1 (SDF1) is important blocks G protein-mediated signaling. CatG-induced upregulation of for an infammatory response (Luster 1998). SDF1 can be MHC I molecules might be a result of interference with the MHC I proteolytically inactivated by cell surface CatG present on B recycling pathway; promoting fusion of the recycling vesicle with the + cell membrane to deliver MHC I molecules to the cell surface. As an cells, NK cells, and ­CD4 T cells (Delgado et al. 2001); this alternative to being degraded or pausing intracellularly, MHC I mole- is of particular interest, since Tregs have the ability to termi- cules can be loaded with a new set of antigenic peptides (viral derived nate an immune response and maintain immune homeosta- or tumor-associated) and pushed back to the cell surface. PBMCs, which carry CatG on the cell surface, as well as the PAR1 antagonist sis (Whiteside 2014). Thus, cell surface CatG might protect (FR171113) drive MHC I to the cell surface, which can be inspected efector cells from friendly fre and supports intercellular by ­CD8+ T cells. CatG-mediated upregulation of cell surface MHC communication between immune cells and target cells via I molecules might be more rapid/efcient by shutting down signal regulation of MHC I. transduction to exert MHC I molecules loaded with a new set of anti- genic peptides, instead provoking the expression of nascent MHC I molecules. Additionally, expression of MHC I is prevented in viral CatG and PARs infected or cancer cells. Our model suggests how CatG circumvents pathogen or cancer cell-derived immune evasion PAR1, PAR2, PAR3, and PAR4 belong to a family of G protein-coupled receptors irreversibly activated by difer- NK subsets, harboring CatG on the cell surface might pro- ent soluble- or cell membrane bound proteases. PARs are teolytically process perforin and granzyme B and thereby substrates for CatG; while CatG proteolytically cleaves the be protected from self-destruction; similar to the proposal N-terminal end of the extracellular domain of PAR1 PAR2, for cathepsin B (Balaji et al. 2002); however, cathepsin and PAR4, unmasking a tethered activation ligand which B-defcient mice did not show impaired CD8­ + T cell sur- fips to the extracellular loop 2, provokes a conformational vival during lysis (Baran et al. 2006). This gives rise to the change and recruits intracellular G protein. G protein is capa- statement that not only one protease might serve as protec- ble of transducing intracellular signal events via an intricate tion from self-lysis, but cell surface CatG might replace the network of downstream signal transduction, as a result regu- self-protective function of cathepsin B. lating, for instance, an infammatory response (Adams et al. An additional functional property of cell surface may be 2011; Heuberger and Schuepbach 2019). Simultaneously, that CatG drives MHC I to the cell surface; when cells are CatG cleavage of the extracellular face of PAR1, PAR2, not infected they will survive, in the case of immune eva- and PAR3 can lead to receptor inactivation (“dis-arming” or sion of viral infected or transformed cells and nascent MHC “of-conformation”) by degrading the tethered ligand or pos- I molecules will not appear on the cell surface after CatG sibly cleaving the three extracellular loops of PAR, thereby engagement. These cells might be recognized by NK cells blocking G protein-mediated signaling (Ramachandran et al. and are activated for killing. 2012). Interestingly, it was shown that pharmacological inhi- What about T regulatory cells (Tregs)? Tregs, which bition of PAR1 reduces the progression of several tumor maintain immune homeostasis and tolerance, are divided types (Flaumenhaft and De Ceunynck 2017), speculating into thymus-derived natural Tregs, which are induced Tregs that inactivated PAR1 in cancer cells might be somehow generated by transforming growth factor (TGF)-β and inter- recognized by immune cells and most likely eliminated by leukin two in vitro, and peripheral resident Tregs (Kanamori CD8­ + T cells. Indeed, we found that exogenous CatG as et al. 2016). A subset of Tregs express the ectonucleotidases well as the PAR1 antagonist (FR171113) enhanced MHC I CD39 ­(CD39+ Tregs) and the CD73 molecule. While CD39 expression on the cell surface of PAR1-expressing PBMCs, hydrolyzes extracellular ATP and ADP towards AMP, CD73 Epstein–Barr virus-transformed B cell line, and glioblas- is in charge for further conversion of AMP generating aden- toma cell line (U87), and sphere-cultured stem cell-enriched osine. Adenosine can then bind to the A2A receptor and cell population from two diferent patients sufering from suppress T cell function (Borsellino et al. 2007; Deaglio glioblastoma (Giese et al. 2016). In the case of glioblastoma et al. 2007; Longhi et al. 2017; Mandapathil et al. 2010). cells, nascent or recycled MHC I bound with tumor-associ- In terms of CD39­ + Tregs, CatG was identifed on the cell ated peptides might be presented and recognized by ­CD8+ T surface by us just recently (Penczek and Burster 2019). cells. This mechanism has to be confrmed experimentally.

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CatG Can Bind to the Cell Surface and is Also evidence that CatG has a more diverse function than previ- Proteolytic Active ously recognized. Indeed, our model suggests how intra- cellular CatG degrades MHC I molecules in the endosomal CatG, by means of a cationic composition due to the high num- compartment or when MHC I molecules pass through the ber of basic amino acids within the CatG protein sequence, recycling pathway. This might sensitize NK cells due to binds to the outer surface of the neutrophil plasma membrane the reduction of cell surface MHC I molecules. On the through electrostatic interactions with the anionic sulfate other hand, exogenous CatG proteolytically cleaves the groups of chondroitin sulfate- and heparan sulfate-containing extracellular domain of PAR1 leading to receptor inacti- proteoglycans (Campbell and Owen 2007; Korkmaz et al. vation and in turn upregulates MHC I molecules. This is 2010). Additionally, CatG has been reported to exhibit binding a mechanism that involves the MHC I recycling pathway to PAR1-4. Like which binds frst to these receptors by supporting the fusion of the recycling vesicle with the and then activates them, CatG might bind to the extracellular cell membrane and thereby delivers MHC I molecules to part of PARs consisting of a negatively charged amino acid the cell surface to present a new set of pathogen-derived residue pattern (Adams et al. 2011). or tumor-associated antigenic peptides. As a result, CatG- Exogenous CatG and NE can bind to the cell surface of can- mediated upregulation of cell surface MHC I molecules cer cells in a specifc and saturable manner and undergo clath- can be inspected by ­CD8+ T and suggests that CatG pre- rin pit-mediated endocytosis (Gregory et al. 2012). According vents pathogen or cancer cell-driven immune evasion. to previous studies, CatG binds to chondroitin sulfate- and heparan sulfate-containing proteoglycans on the cell surface Funding of recipient cells (Campbell and Owen 2007). Interestingly, No funding was received. it was found that treatment of cancer cells with glycanases to Compliance with ethical standards remove respective proteoglycans did not result in diminished cell surface binding or endosomal entry of NE, suggesting Conflict of interest The authors declare no confict of interest. the existence of a specifc cell surface receptor for NE and presumably for CatG, because both CatG and NE share a sig- nifcant sequence identity and they compete for cell surface binding sites. Following binding, CatG enters tumor cells via References endocytosis in a clathrin- and dynamin-dependent manner (fo- tillin-1 and caveolin-1-independent) and apparently process Adams MN, Ramachandran R, Yau MK et al (2011) Structure, func- tumor-derived proteins that might afect tumor cell behavior, in tion and pathophysiology of protease activated receptors. Phar- contrast to NE which induces tumor cell proliferation (Gregory macol Ther 130:248–282 et al. 2012). How can CatG, bonded on the cell surface, still be Adkison AM, Raptis SZ, Kelley DG et al (2002) Dipeptidyl pepti- dase I activates neutrophil-derived serine proteases and regu- active at the site of infammation when natural serine protease lates the development of acute experimental arthritis. J Clin inhibitors are present? 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J Immunol 194:3594–3600 hindrance of natural serine protease inhibitors which cannot Apps R, Del Prete GQ, Chatterjee P et al (2016) HIV-1 Vpu medi- reach the catalytic center when CatG is bonded to the cell ates HLA-C downregulation. Cell Host Microbe 19:686–695 surface (Owen et al. 1995). This allows cell surface CatG to Balaji KN, Schaschke N, Machleidt W et al (2002) Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after be active at the site of infammation even though natural serine degranulation. J Exp Med 196:493–503 protease inhibitors are in direct proximity. Baran K, Ciccone A, Peters C et al (2006) Cytotoxic T lymphocytes from cathepsin B-defcient mice survive normally in vitro and in vivo after encountering and killing target cells. J Biol Chem Conclusion 281:30485–30491 Bartee E, Mansouri M, Hovey Nerenberg BT et al (2004) Downregu- lation of major histocompatibility complex class I by human The summary of our briefng identifed a strong associa- ubiquitin related to viral immune evasion proteins. J tion between the proteolytic activity of CatG and cell sur- Virol 78:1109–1120 face expression of MHC I molecules, indicating a panel of

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